Activation of swelling-activated chloride current by tumor necrosis factor-alpha requires ClC-3-dependent endosomal reactive oxygen production

Abstract

ClC-3 is a Cl(-)/H(+) antiporter required for cytokine-induced intraendosomal reactive oxygen species (ROS) generation by Nox1. ClC-3 current is distinct from the swelling-activated chloride current (ICl(swell)), but overexpression of ClC-3 can activate currents that resemble ICl(swell). Because H(2)O(2) activates ICl(swell) directly, we hypothesized that ClC-3-dependent, endosomal ROS production activates ICl(swell). Whole-cell perforated patch clamp methods were used to record Cl(-) currents in cultured aortic vascular smooth muscle cells from wild type (WT) and ClC-3 null mice. Under isotonic conditions, tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml) activated outwardly rectifying Cl(-) currents with time-dependent inactivation in WT but not ClC-3 null cells. Inhibition by tamoxifen (10 microm) and by hypertonicity (340 mosm) identified them as ICl(swell). ICl(swell) was also activated by H(2)O(2) (500 microm), and the effect of TNF-alpha was completely inhibited by polyethylene glycol-catalase. ClC-3 expression induced ICl(swell) in ClC-3 null cells in the absence of swelling or TNF-alpha, and this effect was also blocked by catalase. ICl(swell) activation by hypotonicity (240 mosm) was only partially inhibited by catalase, and the size of these currents did not differ between WT and ClC-3 null cells. Disruption of endosome trafficking with either mutant Rab5 (S34N) or Rab11 (S25N) inhibited TNF-alpha-mediated activation of ICl(swell). Thrombin also activates ROS production by Nox1 but not in endosomes. Thrombin caused H(2)O(2)-dependent activation of ICl(swell), but this effect was not ClC-3- or Rab5-dependent. Thus, activation of ICl(swell) by TNF-alpha requires ClC-3-dependent endosomal H(2)O(2) production. This demonstrates a functional link between two distinct anion currents, ClC-3 and ICl(swell).

FIGURE 1.

TNF-α activates ICl_swell in WT but not ClC-3 null VSM cells. A, typical currents from WT and ClC-3 null cells ∼10 min after exposure to 10 μm TNF-α. B, I/V plot of the summary data (holding potential −40 mV, test potentials −100 to +100 mV in 20-mV steps; see inset). The response to TNF-α in WT cells is inhibited by either 10 μm tamoxifen (C) or hypertonic (340 mosm) conditions (D). *, significant difference across the full voltage range (p < 0.05). Error bars, S.E.

FIGURE 2.

A, H₂O₂ (500 μm) activates ICl_swell directly in both WT and ClC-3 null cells. Typical current records (above) are paired with I/V plots of peak currents (below). B, scavenging of H₂O₂ with catalase (1000 units/ml) inhibits ICl_swell activation by TNF-α. Error bars, S.E.

FIGURE 3.

Ad-ClC-3 expression in ClC-3 null cells activates ICl_swell even in the absence of cytokine stimulation. This current is further activated by TNF-α, and both responses are inhibited by PEG-catalase. *, difference from non-expressing ClC-3 null cells; #, significant difference from TNF-α. Error bars, S.E.

FIGURE 4.

The magnitude of ICl_swell is unaltered in ClC-3 null VSM cells. Typical current records (above) are paired with I/V plots of peak currents (below). Error bars, S.E.

FIGURE 5.

ICl_swell activation in hypotonic conditions is partially inhibited by PEG-catalase in both WT and ClC-3 null VSM cells. Typical current recordings are shown in A, and I/V summaries for WT (left) and ClC-3 null (right) cells are shown in B. The magnitude of inhibition by catalase is not different between the two groups of cells. *, difference from control or control plus catalase; #, significant difference from hypotonic. Error bars, S.E.

FIGURE 6.

A, Western blot demonstrates expression of S34N Rab5 in cells exposed to adenovirus encoding the protein. Disruption of early endosome sorting with S34N Rab5 prevents activation of ICl_swell by TNF-α but not by hypotonic conditions. B, interfering with endosome recycling using S25N Rab11 also inhibits activation of ICl_swell by TNF-α. Error bars, S.E.

FIGURE 7.

A, thrombin (1 unit/ml) activates ICl_swell in wild type VSM cells. B, a similar current is also activated in ClC-3 null cells, and this current is inhibited by either tamoxifen or catalase. C, S34N Rab5 does not interfere with activation of ICl_swell by thrombin. Error bars, S.E.

FIGURE 8.

Sequential exposure to low pH (4.0), hypotonicity, and finally low pH under hypotonic conditions in WT (A) and ClC-3 null (B) VSM cells reveals that ClC-3 currents (acid-activated (2)) are clearly distinct from those activated under hypotonic conditions. C, when the currents obtained from the WT cell in A under hypotonic conditions at pH 7.35 are subtracted from those observed in the same cell under hypotonic conditions at pH 4.0, the remaining current is very similar to what was initially observed at pH 4.0 under isotonic conditions. Summary data for the magnitude of both peak (D) and late (end of the pulse) currents (E) in WT and ClC-3 null cells confirms that only WT currents are enhanced at low pH. Error bars, S.E.

FIGURE 9.

Schematic comparison of proposed pathways for activation of ICl_swell by TNF-α and thrombin. An activated TNF-α receptor is endocytosed into a Rab5-positive early endosome. This compartment also contains ClC-3 and Nox1 and is the site of TNF-α-mediated ROS production, as previously demonstrated (21). These proteins, including whatever mediates ICl_swell, may become incorporated into the endosome by endocytosis from the plasma membrane (as shown) but could also come from a different source, such as a post-Golgi vesicle. ClC-3-dependent superoxide and subsequently hydrogen peroxide production within this compartment are required for activation of ICl_swell. This could occur by several mechanisms: 1) direct activation via diffusion of H₂O₂ from the endosome to the plasma membrane, 2) direct activation within the early endosome, or 3) indirect activation of ICl_swell via modification of an associated protein or signaling pathway which has the ability to activate ICl_swell (not pictured). The ability of S25N Rab11 to interfere with current activation by TNF-α favors the effect of H₂O₂ on ICl_swell taking place within endosomes. This mechanism is appealing because it compartmentalizes the H₂O₂ required for current activation. Thrombin also signals via Nox1 but does not cause endocytosis (34), suggesting that ROS are produced at the plasma membrane. Thrombin-mediated ICl_swell activation is therefore sensitive to catalase but is not impaired in ClC-3 null cells; nor is it inhibited by S34N Rab5. The dependence of activation of ICl_swell by TNF-α, thrombin, hypotonicity, and ClC-3 overexpression on ClC-3, H₂O₂, and Rab5 are summarized in the inset table. Y, yes; N, no; P, partial.